Bianca Corriea, visiting exchange-student researcher working in the Wyatt Lab, is using antisense RNA (asRNA) to verify the candidate gene responsible for the unique phenotype of the gps3 mutant in response to GPS treatment.
To test the gps3 candidate gene, Corriea must silence the expression of this single gene and verify that this targeted transformation results in the gps 3 phenotype. The gene silencing method that Corriea will deploy is to sequence the candidate gene and then splice a copy of the gene into agrobacterium. Agrobacterium is used in plant biology as a method of introducing genes into the genome of test plants through a process known as agrobacterium-mediated transformation mechanism. This mechanism takes advantage of the agrobacterias natural process of invading host organisms and semi-randomly inserting its DNA into the host genome.
Colin Kruse, Wyatt Lab manager and researcher, is looking for genes not yet identified as related to gravity response in plants. To do this, Colin and Dr. Sarah Wyatt enlisted the help of NASA to germinate seedlings in microgravity conditions aboard the International Space Station. The spaceflown samples have since returned to earth and Colin will soon complete the next milestone in the NASA-sponsored BRIC-20 microgravity experiment. Using RNA extracted from spaceflown seedlings and ground controls, Colin hopes to identify the genes involved in the plant signalling biochemical pathway.
The Wyatt Lab researchers studying the gps2 mutant genome have identified a gene that appears to play a significant role in plant signal transduction. The candidate gene was identified through deep sequencing of the gps2 mutant genome and then comparing the results to the wild type (WT) genome. The analysis revealed a difference of a single gene in one area of the genome associated with plant signaling which had been disrupted by a semi-random T-DNA insertion that silenced (or shut off) the gene in the mutant. In the WT genome, the gps2 gene is intact and expresses normally during GPS treatment. Continue reading
Arabidopsis (common name) gps mutants filmed in time-lapse photography (60 minutes shown) after being returned to room temperature responding to gravity persistent signaling (GPS) treatment.
In the search for genes regulating plant signaling responses to changes in gravity, the Wyatt Lab has focused several research projects on a series of mutant Arabidopsis thaliana plants known as gps mutants. The gps mutants react to gravity differently than wild type (WT) plants. For example, when gps-2 mutants are exposed to the GPS treatment they bend the opposite way from WT plants. The predictable direction each gps mutant strain bends in response to the GPS treatment denotes a phenotype, and these physical differences are important because they are attributable to underlying genetic differences between the plants (that is, differences in genotypes).
Figure 1 – Wild type (here labeled Ws) and gps mutant phenotypes shown at room temperature “remembering” the gravity vector from an earlier GPS treatment. The wild type phenotype (panel A) demonstrates the expected gravitropic reaction; the three gps mutants show aberrant reactions to the GPS treatment characteristic of each phenotype.
One method of determining the role individual genes play in a plant genome is to randomly disrupt the WT genome and observe the effects on the plant. When the WT genome was disrupted through DNA insertion the resulting mutants reacted differently to changes in gravity (gravity persistent signaling) than WT plants. The discovery of gps mutant phenotypes allowed the Wyatt Lab to isolate early signaling events and identify genes controlling plant signal transduction by focusing on genetic differences between WT and gps mutant genotypes in response to the GPS treatment.
Figure 2 – gps2 mutant created from WT through T-DNA insertion of Feldman Tag.
Seven gps mutants have been identified to date, and three gps mutant phenotypes are currently under study at the Wyatt Lab: gps-2, gps-3, and gps-6.